báo cáo khoa học: " Position of the Phi and Po2 loci in the Hal linkage group in pigs" ppsx

pig - Iinkage group - S-Hal-Phi-H-Po2-Pgd - halothane sensitivity - gene order Résumé — La position des loci Phl et Po2 dans le groupe de liaison S, Met Phl, H, PoZ Pgd des porcs..

Original article

Position of the Phi and Po2 loci in the Hal linkage group

in pigs

P Vögeli

Insfifute of Animal Sciences, Breeding Section, Federal Institute of Technology (ETH), CH-8092

Zurich, Switzerland

(received 17 May 1988, accepted 8 October 1988) .

Summary — Families of Swiss Landrace (165 litters with 1348 offspring) were tested for halothane

sensitivity, A-0(S), H, Phi, PGD and Po2 phenotypes Informative matings for the determination of the gene sequence of these linked loci were selected Recombinations were observed between

Phi-Hal, Phi-H and H-Po2 On the basis of these results the most likely order of loci is Hal-Phi-H Confirmation for a locus for genes for Po2 separate from the locus for H is presented The location

of Po2 is between H and Pgd A gene order S-Hal-Phi-H-Po2-Pgd is proposed.

pig - Iinkage group - S-Hal-Phi-H-Po2-Pgd - halothane sensitivity - gene order

Résumé — La position des loci Phl et Po2 dans le groupe de liaison S, Met Phl, H, PoZ Pgd

des porcs Des familles de Porc amélioré suisse (165 portées avec 1348 descendants) ont été

tes-tées pour la sensibilité à 1 halothane, ainsi que pour les phénotypes A-O (S), H, PHI, PGD, et Po2 Des accouplements informatifs pour déterminer l’ordre de ces loci liés ont été sélectionnés Des

recombinaisons ont été trouvées entre Phi-Hal, Phi-H et H-Po2 Ces résultats ont permis de

préci-ser la position du locus Phi dans le groupe de liaison L’ordre le plus probable des loci est

Hal-Phi-H Ces données confirment que les gènes Po2 et H se situent à deux loci distincts Po2 se situe

entre H et Pgd En conclusion est proposé Ibrdre génique S-Hal-Phi-H-Po2-Pgd.

porcs - groupe de Ilaison - S-MeM -H-Po2-Pgd - sensibilité à l’halothane - ordre de loci

Introduction

The linkage between the H blood group locus and the loci for the variants of 6-phospho-gluconate dehydrogenase (Pgtn and phosphohexose isomerase (Phi) was first described

by Andresen (1971 ) Rasmusen & Christian (1976) reported an association between

H genotypes and susceptibility to halothane-induced stress Jorgensen et al (1976) pos-tulated that the association between H and porcine stress and the linkage of Phi and H was the causal link for the association between Phi genotypes and stress susceptibility.

The inheritance of halothane-induced stress has been shown to be controlled by a reces-sive gene at a single locus (Han with incomplete penetrance (Ollivieret al., 1975; Smith &

Bampton, 1977) symbolizes N and n, Hal n Pigs that N/N Nln should there-fore be HAL- non reactors and nln signifies a HALreactor

Linkage studies between Hal and Phi have not made it possible to place the Hal locus

accurately within the linkage group Using a method for calculation of relative linkage

disequilibrium coefficients, Andresen (1979) proposed that Halwas located between Phi and H The gene order Phi-Hal-H-Pgd was also supported by Rasmusen et al (1980).

Their data, however, did not permit them to distinguish between the order Phi-Hal-H-Pgd

as opposed to Hal-Phi-H-Pgd Gu6rin et al (1983) described two recombinants which

supported the order as Hal-Phi-Pgd The recombinants were both HAL- offspring of

matings between Hal Nln and Hal Wn animals The failure of these animals to react to halo-thane could, however, have resulted from the incomplete penetrance of the Haln gene The S locus controls the expression of the A and 0 antigens of the A-O blood group

system in pigs by an epistatic interaction (Rasmusen, 1964 and Hojny & Hdla, 1965).

Two alleles are known, S being dominant over s.

The relationship between A-O blood group phenotypes determined by genes at the S

locus and HAL+ animals (Rasmusen & Christian, 1976) was in agreement with the asso-ciations found between A-O and H blood group systems (Rasmusen, 1972) Hojny (1974) suggested that this association resulted, indirectly, from the genetic linkage between the

H system and the S locus Rasmusen (1981) proposed the order Phi-Hal-S-H-Pgd on the basis of recombinants between S and H as well as between S and Phi-Hal Later, two

.other reports provided evidence that the S locus is not within the Phi-Pgd region, but

adjacent to Phi (Hojny et al., 1985; Van Zeveren et al., 1985)

-Recently it has been found that the serum postalbumin-2 (Po2 locus) also belongs to

the S- (Phi-Hal-H)-Pgd linkage group and is probably located between the H and Pgd

loci (Juneja et al., 1983; Gahne & Juneja, 1985; Cepica et al., 1986).

The aim of this paper is to reconsider the gene order in the linkage group, especially

of the Phi and Po2 loci and to establish the haplotypes (including Hal genotypes) in a

population of Swiss Landrace pigs Estimation of recombination frequencies is given

elsewhere (V6geli et al., 1988).

Materials and Methods

Description of the data

Data for this study came from Swiss Landrace pigs kept at the experimental station of the Institute of Animal Sciences during the period 1983-1988 The total number of offspring

was 1348 The animals came from 165 litters produced by 29 boars and 64 sows over 3

successive generations.

Halothane test

At an age of 8 to 12 weeks the animals were tested for halothane sensitivity by the method of Eikelenboom & Minkema (1974) The anesthetic was a mixture of oxygen and 4% halothane (1.5 liters/min) Negatively reacting animals were exposed for 5 min In

HAL pigs the anesthesia was withdrawn as soon as the symptoms of hyperthermia

(muscular rigidigy, increased heart rate and elevated body temperature) became

appa-rent

Serological tests

The A and 0 reagents were prepared from normal serum of 2 goats and were used in the

hemolytic test The alloimmune anti-Aw was applied in the dextran agglutination test

The blood group factors Ha and Hc were tested using two reagents each One of each

exhibited dosage effects, i.e., they hemolysed red blood cells of homozygous (H / h

H C/ H C ) pigs sooner than those derived from heterozygous (H a / H -, H C / H -) pigs. The validity of the reaction pattern of these reagents was verified in International Pig

Comparison Tests (1984 and 1987, the latter being organized by our laboratory).

Electrophoresis

-The Phi and Pgd phenotypes were determined by horizontal one-dimensional agarose

or starch-gel electrophoresis of hemolysates of erythrocytes (Saison & Giblett, 1969;

Gahne & Juneja, 1985) The Po2 variants were detected by two-dimensional

electropho-resis by the method of Juneja era/ (1983).

Parentage control

Tests for other blood marker systems (B, G, ADA, PGM, P11, P01A, P12 ) were conduc-ted on all animals for the exclusion of incorrect pedigrees.

Haplotyping

The method used in the present study to determine the haplotypes was based on

dedu-cing linkage phases involving Hal and marker loci of both the parents and their offspring.

A detailed description of the procedure is given by V6geli et al (1988) Several instances

of crossing over were observed in progeny from multiheterozygous parents mated to

multihomozygous parents These were used to determine the order of the loci

Results

Table I provides a summary of recombinations involving the Hal and Phi loci recovered in

progeny from matings in which one parent was at least triply heterozygous and the other

doubly or multiply homozygous All the recombinations are informative with respect to the location of the Phi locus The structure of parental haplotypes was inferred from various informative matings.

Assuming that the gene order is Phi-Hal-H as suggested by Rasmusen (1981) and not

Hal-Phi-H, the first 5 recombinants of the first 3 matings given in Table I would have

required the occurrence of a double crossover, i.e., a crossover between Phi and Hal as well as a crossover between Hal and H which is statistically extremely unlikely.

Mating of boar 8888 with female 8849 produced a recombinant (offspring 9925) resul-ting in an unexpected halothane negative reaction of this offspring This recombinant

could be explained as being a result of double crossover However, incomplete

penetran-ce of HaM / HaM seems more likely Unfortunately, the recombinant offspring 9925 was

not saved for breeding to determine his actual genotype In the offspring of animals with Hafn Hain genotype mated to Ha! / HaM, about 10% are classified as HAL- (Gahne &

Juneja, 1985.) The failure of one offspring from a total of six to react to halothane could well be the result of incomplete penetrance of the Hal gene From these considerations the gene order of Hal-Phi-H is suggested.

informative recombinants between S, Hal, Phi and H on side and Po2 and Pgd on the other All five recombinants are informative in determining the

position of the Po2 locus From these data the gene order is H-Po2-Pgd as proposed by

Juneja et al (1983) If the gene order were Po2-H-Pgd, all 5 recombinants could only

have resulted from double crossovers (Phi Î-P 2 Î-H-Pgd), which is highly improbable.

Table III shows the parents and offspring of 2 litters which include recombinants

invol-ving a crossover between loci for Phi and H types These marker loci are also consistent with a gene order of Phi-H-Po2 as opposed to H-Phi-Po2

Discussion

The expected Hal genotype of offspring receiving (a) recombinant haplotype (s) can be determined if the sequence between Hal and marker loci has been established The

most likely order of the marker loci including Hal was indicated as S-(Phi-Hal) -(H-Po2)-Pgd by Hojny et al (1985) and van Zeveren et al (1985) As these authors did not detect

crossing over between Phi and Hal, they could neither prove nor disprove the reverse

sequence for the Phi and Hal loci proposed by Gu6rin et al (1983) However, they confir-med that the two loci are located very close to each other

The most important contribution of this paper is the evidence that the Phi locus is

located, most probably, between Hal and H as proposed by Guérin et al (1983) and van Zeveren et al (1988) and not between S and Hal as previously reported by Andresen

(1981) and Rasmusen (1981) This location is more firmly established by complex

S-Hal-Phi-H-Po2-Pgd haplotypes of the majority of parents and offspring, including recom-binants Probably because of incomplete penetrance of the Hal gene one animal with

presumed genotype Hah / HaM failed to react to halothane Two recombinants (Table 1,

offspring 695) being respect to the location of the Phi locus were classified as HAL These two reactors certainly are Hal !l Hal ! homozygotes

because the probability of a Hah l HaI or HalN / Hain pig being falsely tested as HAL is very low (V6geli et al., 1988).

The data in Tables II and III are consistent with a gene order of Phi-H-Po2-Pgd The

data assembled in Tables I, II and III and those contained in earlier publications indicate

a gene order S-Hal-Phi-H-Po2-Pgd The knowledge of the halothane locus and its

linka-ge relationships is already being used in practical animal breeding to reduce the

frequen-cy of the Hal ! gene (Gahne & Juneja, 1985; V6geli et al., 1988) Looking to the future,

molecular analysis of the halothane linkage group may provide a means for identifying

more reliable markers for the stress genes as well as the identity of the halothane gene itself One step in this development is the assignment of the Hal linkage group to chro-mosome 6 by in situ hybridization (Davies ef al., 1988).

Acknowledgements

This work was supported by grants of the ETH-Zurich, the Commission for Support of Scientific

Research, Berne, the Swiss Performance Testing Station, Sempach, and other public and private organizations in Switzerland Appreciation is expressed to my coworkers Christine Karonis, Barbara Kuhn and R Kuhne for excellent technical assistance and Drs N.S Fechheimer, Valerie Madison,

Catherine Marguerat and G Stranzinger for comments on the manuscript Data collection on the

experimental farm are made possible by Dr C Gerwig and A Kaufmann.

Andresen E (1971) Linear sequence of the autosomal loci PHI, H and 6-PGD in pigs Anim Blood

Groups Biochem Genet 2, 119-120

Andresen E (1979) Evidence indicating the sequence Phi, Hal, H of the three dosely linked loci in

pigs Nord Veterinaermeal 31, 443-444

Andresen E (1981) Evidence for a five-locus linkage group involving direct and associative

interac-tions with the A-O blood group locus in pigs In : (E Brummerstedt ed.), Papers dedicated to Prof.

Dr J Moustgaard, 208-212 The Royal Danish Agricultural Society, Copenhagen.

!epica S., Hradecki J., Hojny J., Kuryl J & Grzybowski G (1986) Localization of the Po2 locus in the S, Phi, Hal, H, Po2, Pgd linkage group in pigs Anim Genet 17, 283-286

Davies W., Harbitz L, Fries R., Stranzinger G & Hauge J.G (1988) Porcine malignant hyperthermia carrier detection and chromosomal assignment using a linked probe Anim Genet 19, 203-212 2

Eikelenboom G & Minkema D (1974) Prediction of pale, soft, exudative muscle with a non-lethal

test for the halothane-induced porcine malignant hyperthermia syndrome Tijdschr Diergeneesk 99,

421-426

Gahne B & Juneja R.K (1985) Prediction of the halothane (Hal ) genotypes of pigs by deducing

Hal, Phi, Po2, Pgd haplotypes of parents and offspring: results from a large-scale practice in

Swedish breeds Anim Blood Groups Biochem Genet 16, 265-283

Gu6rin G., Ollivier L & Sellier P (1983) Etude de groupe de liaison Hal, Phi et Pgd chez le Porc:

disposition relative des trois locus et estimation des taux de recombinaison Genet Sel Evol 15,

55-64

Hojny J (1974) H blood group genotypes and expression of A and 0 antigens in pigs Anim.

Blood Groups Biochem Genet 5, 3-10 0

Hojny J & Hila K (1965) A contribution to the study of the blood group system A in pigs In: Blood groups of animals (J Matousek ed.) Publishing House of the Czechoslovak Academy of Sciences,

Prague pp.155-161

Hojny, Cepica S & Hradecky J (1985) Gene order and recombination rates in the linkage group

S-Phi-Hal-H-(Po2)-Pgd in pigs Anim Blood Groups Biochem Genet 16, 307-318 8

Juneja R.K., Gahne B., Edfors-Lilja I & Andresen E (1983) Genetic variation at a pig serum protein

locus, Po-2 and its assignment to the Phi, Hal, S, H, Pgd linkage group Anim Blood Groups

Bio-chem Genet 14, 27-36

Jorgensen P.F., Hyldgaard-Jensen J., Moustgaard J & Eikelenboom G (1976) Phosphohexose

iso-merase (PHI) and porcine halothane sensitivity Acta Vet Scand 17, 370-372

Ollivier L., Sellier P & Monin G (1975) D6terminisme g6n6fique du syndrome d’hyperthermie maligne chez le porc de Pi6train Ann Genet Sél Anim 7,159-166

Rasmusen B.A (1964) Gene interaction and the A-O blood group system in pigs Genetics 50,

191-198

Rasmusen B.A (1972) Gene interaction and the A-O and H blood group systems in pigs Anim Blood Groups Biochem Genet 3, 169-172

Rasmusen B.A (1981) Linkage of genes for PHI, halothane sensitivity, A-O inhibition, H red blood cell antigens and 6-PGD variants in pigs Anim Blood Groups Biochem Genet 12, 207-209

Rasmusen B.A & Christian L.L (1976) H blood types in pigs as predictors of stress susceptibility.

Science 119, 947-948

Rasmusen B.A., Beece C.K & Christian L.L (1980) Halothane sensitivity and linkage of genes for

H red blood cell antigens, phosphohexose isomerase (PHI) and 6-phosphogluconate

dehydrogena-se (6-PGD) variants in pigs Anim Blood Groups Biochem Genet 11, 93-107

Saison R & Giblett E.R (1969) 6-phosphogluconic dehydrogenase polymorphism in the pig. Vox Sanguinis 16, 514-516 6

Smith C & Bampton PR (1977) Inheritance of reaction to halothane anaesthesia in pigs Genet Res

Van Zeveren A., Weghe A., Bouquet Varewyck (1985) position epistatic

locus in the halothane linkage group in pigs Mim Blood Groups Biochem Genet.16, 297-305

Van Zeveren A., Van de Weghe A., Bouquet Y & Varewyck H (1988) The porcine stress linkage

group 11 The position of the Halothane locus and the accuracy of the Halothane test diagnosis in Belgian Landrace pigs J Anim Breed Genet 105, 187-194

’

Vögeli P., Gerwig C & Schneebeli H (1983) The A-O and H blood group systems, some enzyme

systems and halothane sensitivity of two divergent lines of Landrace pigs using index selection

pro-cedures Livest Prod Sci 10, 159-169

Vtigeli P., Kuhue R., Gerwig C., Kaufmann A., Wysshaar M & Stranzinger G (1988) Bestimmung

des Halothangenotyps (Hal ) mit Hilfe der S, Phi, Hal, H, Po2, Pgd Haplotypen von Eltem und Nachkommen beim Schweizerischen Veredelten Landschwein Z chtungskunde 60, 24-37